本文選題：肺炎鏈球菌 + 細(xì)菌粘附　； 參考：《重慶醫(yī)科大學(xué)》2009年博士論文

【摘要】： 肺炎鏈球菌(Streptococcus pneumoniae, S.pn)是一種常見的革蘭陽性條件致病菌,可引起肺炎、中耳炎、菌血癥、腦膜炎等嚴(yán)重疾病,在全球有很高的發(fā)病率和死亡率。老人、兒童、艾滋病和先天性免疫缺陷的患者是S.pn感染的高危人群。由于S.pn抗生素耐藥率的增加和目前疫苗的缺陷性,使得其防治面臨困境。要想從根本上解決S.pn的防治問題,就要深入了解其致病的分子機(jī)制,為研發(fā)新的藥物和疫苗提供理論和實驗依據(jù)。 HSP100/ ClpATPase是一類高度保守并廣泛存在的熱休克蛋白(heat shock protein, HSP),它除了具有分子伴侶的功能外,還與肽酶ClpP形成ATP依賴的蛋白酶復(fù)合物(如ClpAP,ClpBP等)特異性水解某些蛋白質(zhì)。研究顯示ClpATPase可影響某些基因的轉(zhuǎn)錄因子活性,從而調(diào)控包括毒力因子在內(nèi)的多種基因的表達(dá),導(dǎo)致細(xì)菌生長、毒力等多種生物學(xué)性狀發(fā)生改變。肺炎鏈球菌有四種ClpATPase(ClpC,ClpE,ClpL,ClpX)。已有的研究顯示ClpC、ClpL及ClpX都與S.pn的感染致病相關(guān)。但ClpE目前僅知道是肺炎鏈球菌溫度耐受的主要元件,對于其在肺炎鏈球菌致病過程中的作用尚不清楚。 本課題從整體水平、細(xì)胞水平和分子水平初步探索了ClpE在肺炎鏈球菌致病過程中的作用及機(jī)制,這可為深入了解肺炎鏈球菌致病機(jī)制提供理論和實驗依據(jù)。本課題包括以下兩個部分內(nèi)容: 1、構(gòu)建肺炎鏈球菌clpE缺失突變體,比較clpE缺陷菌株和野生菌D39對小鼠的毒力差異,從總體水平上初步了解ClpE是否參與了肺炎鏈球菌的致病。 采用長臂同源多聚酶鏈?zhǔn)椒磻?yīng)(Long flanking homology polymerase chain reaction,LFH-PCR)方法,制備中間為紅霉素耐藥基因(erm)兩側(cè)為clpE基因上、下游同源序列的連接片段,并將此片段轉(zhuǎn)化入肺炎鏈球菌D39,在含紅霉素的血平板上篩選出clpE缺陷菌株,并通過PCR、測序進(jìn)行鑒定;小鼠體內(nèi)實驗比較clpE缺陷菌株和野生菌D39對小鼠的毒力差異。PCR和測序結(jié)果顯示,我們構(gòu)建D39clpE缺陷菌株成功;小鼠體內(nèi)實驗結(jié)果顯示,clpE缺陷菌株對小鼠的毒力較野生菌株明顯減低(P0.01)。研究結(jié)果提示,ClpE可能在肺炎鏈球菌的致病過程中發(fā)揮了作用。 2、從細(xì)胞水平、分子水平進(jìn)一步了解ClpE在肺炎鏈球菌致病過程中的作用及其機(jī)制。 本研究觀察了不同溫度下clpE缺陷菌株和野生菌株在液體培養(yǎng)基中的生長情況;體外粘附、侵襲實驗比較缺陷菌株與野生菌株對宿主細(xì)胞(人肺腺癌細(xì)胞A549、人臍靜脈內(nèi)皮細(xì)胞HUVEC)的粘附、侵襲能力;熒光定量PCR檢測肺炎鏈球菌幾種主要毒力因子在缺陷菌株和野生菌株中的表達(dá)差異;雙向凝膠電泳比較缺陷菌株和野生菌株的蛋白表達(dá)差異,借助基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜(MALDI-TOF-MS)對部分蛋白質(zhì)分子進(jìn)行分析鑒定,并結(jié)合生物信息學(xué)方法尋找ClpE調(diào)節(jié)的蛋白質(zhì)分子。 結(jié)果顯示:1、在37℃時,clpE缺陷菌的生長繁殖能力較野生菌株大大降低,而在40℃、42℃時,缺陷菌幾乎不能生長。2、clpE缺陷菌株對宿主細(xì)胞的粘附和侵襲能力都顯著弱于相應(yīng)的野生菌株(P0.05)。3、同野生菌株比較,幾種重要毒力基因自溶素(major autolysin A,lytA)、表面粘附素A(pneumococcal surface adhesion A,psaA)、溶血素(pneumolysin,ply)、神經(jīng)酰胺酶(neuraminidase, nanA)和鏈球菌表面蛋白A(pneumococcal surface protein A, pspA)在clpE缺陷菌中的表達(dá)量顯著下降(P0.05)。4、與細(xì)菌生長、粘附侵襲、生物被膜形成密切相關(guān)的蛋白質(zhì)如次黃嘌呤-鳥嘌呤磷酸核糖轉(zhuǎn)移酶(Hypoxanthine-guanine phosphoribosyltransferase)、甲酸-四氫葉酸酯連接酶( Formate-tetrahydrofolate ligase )、吡咯烷羧酸肽酶(Pyrrolidone-carboxylate peptidase 1)和雙功能蛋白PyrR (bifunctional protein PyrR)在缺陷菌中的表達(dá)量均明顯低于其野生菌。這些結(jié)果提示ClpE可能通過調(diào)控包括毒力因子在內(nèi)的一些蛋白質(zhì)的表達(dá)來影響肺炎鏈球菌的生存、對宿主細(xì)胞粘附、侵襲等多種生物學(xué)功能。 總之,本研究顯示ClpE可通過調(diào)控多種蛋白質(zhì)的表達(dá)來影響細(xì)菌的生存能力、對宿主細(xì)胞的粘附侵襲能力以及對宿主細(xì)胞的損傷能力,它是肺炎鏈球菌在宿主體內(nèi)存活并導(dǎo)致感染發(fā)生所必須的。
[Abstract]:Streptococcus pneumoniae ( S.pn ) is a common pathogen of Gram - positive conditions , which can cause severe diseases such as pneumonia , otitis media , baccaemia , meningitis and other serious diseases , and has a high incidence and mortality rate in the world .



HSP100 / ClpATPase is a highly conserved and widely - existing heat shock protein ( HSP ) , which specifically hydrolyzes certain proteins in addition to the function of molecular partners . It also specifically hydrolyzes certain proteins with peptidase ClpP . The research shows that ClpATPase can affect the transcription factor activity of certain genes , thus regulating the expression of various genes including virulence factors , resulting in changes in various biological properties including virulence factors . There are four kinds of ClpATPase ( ClPC , ClpE , ClpL , ClpX ) . It has been shown that ClPC , ClpL and ClpX are all related to the infection of S.pn . However , ClpE is only known to be the main component of temperature tolerance of Streptococcus pneumoniae , which is not known for its role in the pathogenesis of Streptococcus pneumoniae .



The role and mechanism of ClpE in the pathogenesis of Streptococcus pneumoniae were investigated from the overall level , cell level and molecular level , which could provide a theoretical and experimental basis for further understanding the pathogenesis of Streptococcus pneumoniae .



1 . To construct the deletion mutant of Streptococcus pneumoniae clpE , compare the virulence difference between clpE - deficient strain and wild strain D39 , and preliminarily understand whether ClpE is involved in the pathogenesis of Streptococcus pneumoniae .



Using long - arm homologous polymerase chain reaction ( LFH - PCR ) method , the clpE gene on both sides of erythromycin - resistant gene ( erm ) was prepared . The fragment was transformed into Streptococcus pneumoniae D39 . The strain of clpE - deficient strain was screened by PCR and sequencing . The results of PCR and sequencing showed that the strain of clpE - deficient strain was significantly lower in mice than wild strain ( P0.01 ) . The results suggested that ClpE could play a role in the pathogenesis of Streptococcus pneumoniae .



2 . The role and mechanism of ClpE in the pathogenesis of Streptococcus pneumoniae were further investigated from cell level and molecular level .



The growth of clpE - deficient strain and wild strain in liquid medium was observed under different temperatures . The expression of several major virulence factors in the host cell ( human lung adenocarcinoma cell A549 , human umbilical vein endothelial cell HUVEC ) was compared with wild strain in vitro adhesion , invasion experiment and fluorescence quantitative polymerase chain reaction ( PCR ) . The difference of protein expression between the defective strain and wild strain was detected by fluorescence quantitative PCR , and the protein molecule was analyzed and identified by MALDI - TOF - MS by means of matrix assisted laser desorption ionization time - of - flight mass spectrometry ( MALDI - TOF - MS ) .



The results showed that : 1 . The growth and reproduction ability of clpE - deficient strain was significantly lower than that of wild strain at 37 鈩，

本文編號：1771288